The goal of the proposed research is to use a combined genetic/genomic approach in the fission yeast Schizosaccharomyces pombe to understand the roles of exonic splicing enhancers and the proteins that recognize and respond to them. Experiments in mammalian cell extracts have led to a model in which enhancer-bound SR proteins contact the heterodimeric general splicing factor U2AF and stabilize its association with weak 3' splice sites. However, the relevance of this model to splicing in vivo has not been established. S. pombe is the simplest eukaryote that contains highly conserved orthologues of proposed enhancer complex constituents. Thus, the availability of facile genetic tools and a complete genome sequence will be exploited to extend current knowledge of these important signals and factors under three specific aims: 1) a) The role of an exonic splicing enhancer in promoting and/or regulating splicing of srp2 pre-mRNA, which encodes the fission yeast counterpart of human SRp55, will be delineated using a battery of molecular genetic manipulations followed by assays of splicing in vivo. b) Other naturally occurring S. pombe exonic splicing enhancers will be sought using a gene fusion strategy and their source genes identified. 2) a) To determine whether Srp2p functions as a general or specialized splicing factor in fission yeast, the pattern of splicing defects for a diverse panel of introns will be analyzed after physically or genetically depleting the protein in vivo. b) The roles of both subunits of U2AF in enhancer-dependent vs. enhancer-independent splicing will be determined by assaying splicing of selected pre-mRNAs in cells harboring conditional mutations. 3) Both open-ended and directed genetic strategies will be employed to identify other genes whose products physically or functionally interact with fission yeast SR proteins and with the two subunits of U2AF. Identification and characterization of these components should not only provide important new information about contacts between components known to interact, but reveal novel factors that collaborate with SR proteins and U2AF within the cell. Together, these studies should not only shed new light on specific questions about the early events of pre-messenger RNA splicing, but lead to a more global picture of their impact on the cell.